Mycobacterium tuberculosis (M. tuberculosis) causes tuberculosis (TB). Each year, about 3 million people around the world are suffering from M. tuberculosis induced TB. China is one of the 22 countries around the world that have relatively high level of TB infections. In fact, following India, China has the second largest population of TB patients in the world. Recently, the number of people infected by TB in China is increasing again. According to the 4th nationwide TB epidemiological survey in 2000, China was rated high in several aspects: including the number of people infected (there are up to 550 million people infected, with the infection rate of 45%, which is significantly higher than the average infection level worldwide), the number of patients (there are about 5 million patients with active lung TB), the number of new cases (there are about 1.5 million new cases of active lung TB per year), the fetal rate (which is about 0.2 million per year), and the number of drug-resistant patients (which is around 30% of the patients are drug resistant). We can see from the aforesaid data that the world, especially China, should put in tremendous and imperative effort to control the spread of TB.
TB is an infectious respiratory disease, which is best prevented and controlled by vaccinations. Bacille Calmette Guerin (BCG) is the only TB vaccine so far. BCG is an attenuated vaccine that was discovered by French doctors Calmette and Guerin in 1921. Calmette and Guerin had cultivated 230 generations of M. bovis in 13 years before they got an attenuated vaccine successfully. The TB vaccine was named after Calmette and Guerin to memorize their great contributions. Since the technique of reserving bacteria in freezers has yet been developed back then, said bacteria were grown on slanted surface. With other reasons like wars, the originally cultivated strain may have already been lost. Nowadays, several strains are used for BCG production in the world. The one used in China is a strain from Denmark. Vaccinating newborn babies with BCG can prevent severe pediatric TB, like tuberculosis meningitis, and acute military tuberculosis. However, the preventive effect of BCG has not been confirmed (Baily G V, et al, Ind. J. Med. Res., 72:1-74, 1980). Moreover, BCG is useless in protecting those who have already been exposed to Mycobacteria in the environment, or who have already been infected by M. tuberculosis from TB (Brandt L., et al. Infect. Immun., 70:672-678, 2002). Due to the aforesaid reasons, China has abolished adult BCG vaccination in 2000. To improve the diagnosis of TB, countries with low incidences of TB no longer vaccinate newborn babies with BCG. Since the BCG is an attenuated vaccine, it can not be used to prevent TB from those, such as AIDS patients, who have compromised immune systems, while it is easier for those with compromised immune systems to be infected by M. tuberculosis. 
In the late 1980s, the number of cases of TB in the United States appeared to be going up again. The international attention was then directed to researches on new TB vaccines, and WHO has founded a TB vaccine research team at the end of last century.
China is an area with relatively large number of TB patients: around 550 million people are infected by M. tuberculosis, 10% out of which will develop into TB. By preventing the M. tuberculosis infected population from developing into TB, incidences of TB can be lowered, and the control of TB can be achieved more effectively. The high risk population always uses oral Isoniazid for prevention; however, it is usually hard to stick to as patients need to take it for a long time. Moreover, Isoniazid often causes adverse reactions like peripheral neuropathy, and liver damage, as well as special adverse reactions, such as epilepsy, psychonosema, autonomis nervous disorders, etc (Guo L, et al. Chinese Journal of Coal Industry Medicine, 3(9): 940-941, 2000). Therefore, the high risk population needs to be vaccinated for TB prevention, as it is easy, convenient and with little side effects.
Reported TB vaccines can be divided into two general categories: live bacteria vaccine (including live attenuated vaccine, and recombinant BCG) and non-bacteria vaccine (including protein unit vaccine and DNA vaccine). Attenuated vaccines are normally made from bacteria strains that have nutrient defects, for example a strain that has defect in leucine synthesis. The reported fadD26 is a genetically mutated strain of M. tuberculosis. The gene product of fadD26 is a chemical similar to acetyl coenzyme A, and it is in charge of phthioceral dimycocerosates synthesis. Mutations of fadD26 will attenuate M. tuberculosis (Infante E, et al. Clinical and Experimental Immunology, 141:21-28, 2005). Since attenuated bacteria strains have the potential of mutating back to its origins (Sampson S L, et al, Infect. Immun., 72:3031-3037, 2004), it is risky to vaccinate those who have compromised immune systems with live attenuated bacteria vaccines. Live attenuated bacteria vaccines may also resemble BCG's fate, namely useless in protecting the infected population. Once DNA vaccines are discovered, since it can stimulate cell immunity, the search for a TB DNA vaccine has become an increasingly hot research topic, and a number of articles have reported DNA vaccines (Sugawara I, et al., Tuberculosis. 83:331-337, 2003). In the process of vaccination by DNA vaccines, encoding genes are incorporated into eukaryotic vectors to construct expression vectors of eukaryotic cell, then using E. coli to do plasmid amplification. After the recombined expression vectors of DNA are extracted and strictly purified, naked DNA is injected into muscles for immunity. Various research groups have found that DNA vaccines work safer, more reliable, and better with protein subunit vaccines.
The choice of antigen is absolutely essential in the research of DNA vaccines and protein subunit vaccines. The use of a single kind of antigen does not give ideal results on protecting animals, which are infected by M. tuberculosis, from TB, while multiple single proteins or fusion proteins work better than a single kind of antigen. So far, it has been reported that combined antigens, which are cocktails of fusion proteins and multiple antigens, DNA form, recombined proteins, or secreted proteins from the filtered culture of M. tuberculosis (Roberts A. D. et al, Immunology, 85: 502-508, 1995). It has also been reported that if protein Ag85B(Rvv1886c) and protein ESAT-6(Rv3875) are mixed in the ratio of 1:1 to immune mice, it gives better result than using protein ESAT-6 alone, but it gives worse result than using protein Ag85B alone or using fusion protein Ag85B-EAST-6 (Olsen A W, et al, Infect. Immun., 72:6148-6150, 2004); Ag85B DNA vaccines, MPT64(Rv1980c) DNA vaccines and MPT83(Rv2873) DNA vaccines mixed in the ratio of 1:1:1 to immune cows (Cal H, et al, Vaccine, 23:3887-3895, 2005). If comparing between a single recombinant antigen with fusion protein, as mentioned above, fusion protein has better results than mixing single antigens. Moreover, from the mass production point of view, it is more economical and simple to produce one fusion protein than two recombinant proteins simultaneously. During the past few years, fusion proteins were reported to be used in the research of DNA vaccines, protein subunit vaccines, and recombinant BCG. Fusion proteins mainly include: Ag85B-ESAT-6, ESAT-6-Ab85B, and Mtb72F(mtb39-Ag85B) (Langermans JAM, at al. Vaccine, 23:2740-2750, 2005, Olsen A W, et al, Infect. Immun., 69: 2773-2778, 2001).
The number of drug-resistant patients in China is huge, which amounts to 30% of the total number of patients. In the past 40 years, there has been no novel TB chemotherapeutic drug on the market; and chemotherapies are facing the problem of consistently high rate of drug resistance. One possible solution to treat drug-resistant patients or hard-to-treat TB patients is to use immuno-therapies to improve chemotherapies. So far, main TB immuno-therapeutic drugs for clinical uses include thymopolypeptides for injection and freeze-dried M. Vaccae Vaccine for Therapy-Vaccae. Thymopolypeptides give adverse effects like febris and fever. Non-bacterial vaccine “Vaccae”, which is prepared by rupturing M. Vaccae cells, has been put onto the market since 2001. It is used for TB immunity treatments mainly, it can also be used to prevent or treat M. tuberculosis infections.
Currently, it is urgent to further develop TB vaccines, which have high immunogenicity and small adverse effects, and which can be used for TB immunity treatments. The present invention provides a fusion protein, the expression level of which is 33-38% out of all expressed bacterium proteins. This percentage is significantly higher than the expression level of other current recombinant M. tuberculosis fusion proteins in the field, and will fully satisfy the clinical demand. Furthermore, the present invention can significantly stimulate immuno-response directly against M. tuberculosis; therefore, it can be used as an antigen to prevent, treat, or diagnose TB, as well as to conduct epidemiological survey and monitoring. The present M. tuberculosis fusion protein can be used to prepare new vaccines which can substitute BCG, and therefore provide a new option to prevent or treat TB.